It is well known that human milk-feeding is considered superior to formula-feeding for infants. Not only does human milk provide a well-balanced supply of nutrients, but it is also easily digested by the infant. Thus, several biologically active components which are known to have physiological functions in the infant are either a constituent of human milk or produced during the digestion thereof, including components involved in the defense against infection and components facilitating the uptake of nutrients from human milk.
In spite of the great efforts which have been invested in preparing infant formulae, it has not been possible to produce a formula which to substantial extent has all the advantageous properties of human milk. Thus, infant formula, often prepared on the basis of cow milk, is generally incompletely digested by the infant and is lacking substances known to have effect on the physiological functions of the infant. In order to obtain an infant formula with a nutritional value similar to human milk, a number of additives including proteins, protein fragments, vitamins, minerals etc., which are normally formed or taken up during the infant's digestion of human milk, are included in the formula with the consequent risk of posing an increased strain on and possible long-term damage of important organs such as liver and kidney. Another disadvantage associated with the use of cow milk-based formulae is the increased risk for inducing allergy in the infant against bovine proteins.
As an alternative to cow milk-based infant formulae, human milk obtainable from so-called milk banks has been used. However, feeding newborn infants with human milk from milk banks has in the recent years to an increasing extent been avoided, because of the fear for the presence of infective agents such as HIV and CMV in human milk. In order to destroy the infective agents in human milk it has become necessary to pasteurize the milk before use. However, by pasteurization the nutritional value and the biological effects of the milk components are decreased or abolished. Hence, human milk is used to a still lesser extent.
Presently, commercially available human infant formula used to replace mother's milk is based primarily upon the protein constituents of cow's milk. These infant formula compositions have led to difficulties in terms of nutrient balance, bio-availability of nutrients and sensitivity of human infants to non-human/animal protein. Specifically, allergic reactions to the non-human animal protein used with these infant formulas caused a change in the protein component of the commercially available formula to soy-protein based formulas, although many infants that are allergic to cow's milk are also allergic to soy-based milks (Am. Acad. of Pediatrics Comm. on Nutrition, Pediatrics 72, 359-363 (1983)).
Additionally, many of the problems with the use of cow's milk protein are associated with difficulties in digestibility because of bovine casein content and structure (L. Hambraeus, E. Forsum and B. Lonnerdal. In: "Food and Immunology", pp. 116-124 (Eds. L. Hambraeus, L. A. Hanson and H. McFarlane) Almquist and Wiksell (1977)).
This has led to the production of infant formulas which contain a greater proportion of whey protein, since it is more readily digested by human infants (M. J. Newport and M. J. Henschel, Pediatric Res. 18, 658-662 (1984)), and little or no bovine casein. However, the major protein in whey of cow's milk is .beta.-lactoglobulin. This protein is essentially absent from human milk and has been determined to be one of the main causes of cow's milk allergy in infants (I. Axelsson, I. Jakobsson, T. Lindberg and B. Benediktsson, Acta Pediatrica Scand. 75, 702-707 (1986)). The extent of the problems with allergies to formulas based on cow's milk may be appreciated from the fact that soy-based formulas now comprise a large portion of the human infant formula market in the United States.
Soy-protein formulas, although different in carbohydrate and protein source, are similar in composition to cow's milk protein formulas following the American Academy of Pediatrics, Committee on Nutrition recommendations for nutrient levels in infant formulas. Differences include a slightly higher protein level and slightly lower carbohydrate content. The protein source is generally soy-protein; the fat is a blend of vegetable oils; and the source of carbohydrate is usually sucrose, corn syrup solids, or a mixture of both. However, the use of soy formulas tends to raise serum alkaline phosphatase and blood urea levels in infants in addition to causing the allergic and digestibility problems encountered with the use of bovine-based protein infant formulas.
Human milk differs markedly from that of other mammalian species, including cows, in that it contains a lower over-all protein content and lower ratio of casein/whey as well as a different protein composition. For instance, the casein subclasses of human milk comprises only .beta.-casein and .kappa.-casein, whereas the bovine casein subclasses are .alpha.-casein, .beta.-casein, and .kappa.-casein (Miller et al. 1990). Also the amino acid compositions of human milk protein differ from that of other mammalian milk proteins.
.kappa.-casein is a glycosylated protein which is present in milk of several species including man. Human .kappa.-casein has been shown to contain several (up to 10) prosthetic sugar groups distributed throughout the peptide chain instead of 0-5 as in the case of cow and sheep .kappa.-casein.
A number of different biological activities have been suggested for .kappa.-casein and .kappa.-casein peptides. For a review see e.g. Miller et al. 1990 and Fiat and Jolles 1989. .kappa.-casein has been shown to have a calcium binding site (Fitzgerald and Swaisgood, 1989). Examples of other functions of .kappa.-casein or fragments thereof released during digestion are inhibition of gastrin secretion and thus acid secretion in the stomach (Stan et al, 1982), a regulatory effect on gastrointestinal hormones and thus on release of enzymes from exocrine pancreas (Yvon et al, 1987), growth promoting effects on Lacto-bacillus bifidus pennsylvanicus (Bezkorovainy et al, 1979) and Bifidus infantis (Azuma et al, 1984), opioid-antagonist activity (Chiba et al., 1989), inhibition of angiotensin 1-converting enzyme (ACE) (Marayama et al, 1987), inhibition of platelet aggregation (Jolles et al., 1986), immunostimulatory properties (Jolles et al, 1982) and various antimicrobial effects (Miller et al, 1990). A digestion product (.kappa.-caseino-glycopeptide) of human .kappa.-casein has been found to inhibit the adhesion of certain bacteria, Streptococcus pneumoniae and Haemophilus influenzae, to human respiratory tract epithelial cells (Aniansson et al. 1990).
It would be desirable to be able to prepare an infant formula with a composition closer to that of human milk and thus avoid the above disadvantages associated with bovine milk-based infant formula, e.g. a formula comprising human milk proteins. However, this would require that human milk proteins are obtainable in large quantities. Although human milk proteins may be purified directly from human milk, this is not a realistic and sufficiently economical way to obtain the large quantities needed for large scale formula production, and other methods must be developed before an infant formula comprising human milk proteins may be prepared.
Chobert et al. in 1976 isolated the so called caseinomacropeptide, the glycosylated C-terminal part of human .kappa.-casein, and determined part of its amino acid sequence. The complete sequence of the C-terminal part of human .kappa.-casein was determined by Fiat et al. 1980. The sequence of the para-.kappa.-casein, the N-terminal part of .kappa.-casein, was later determined by Brignon et al. 1985. The complete sequence of the native human .kappa.-casein was reported to contain 158 amino acids.
Several milk protein genes, primarily from rodents or dairy animals, have been cloned and sequenced, but knowledge of the genes encoding human milk proteins is still sparse. Hall et al. 1987 published the sequence of the human .alpha.-lactalbumin gene. Menon and Ham 1989 disclosed the isolation and sequencing of a partial cDNA clone encoding human .beta.-casein, the complete cDNA sequence was later determined by Lonnerdal et al. 1990. In WO91/08675 is described human infant formulas containing recombinant human .alpha.-lactalbumin and .beta.-casein. The sequence of the human lactoferrin cDNA was published by Powell and Ogden 1990. The cDNA cloning of human milk bile salt-stimulated lipase was published by Nilsson et al. 1990. Menon et al., 1991 disclosed a mRNA from which a part of amino acid sequence (3'-end) of the human .kappa.-casein can be deduced.